Microorganisms are being widely used as sources of useful chemical products, whether naturally occurring or through genetic engineering. Screening of organisms, particularly single cell organisms, is necessary for identifying those that produce useful products or useful products in more abundant quantities. Such identified organisms may then be isolated and utilized for their useful attributes.
Traditionally, screening of a population of organisms has been performed on agar plates. The population is usually spread on plates containing an indicator for a target enzyme, for example a chromogenic substrate, or skim milk to detect proteases. Colonies of the plated organism which produce elevated levels of the target enzyme can be detected, based on the intensity of color, the clearing zone, etc.
The method of screening by plating on agar plates has several disadvantages. The conditions on agar plates are very different from production conditions, which generally involve high-density fermentation. Therefore, a strain of an organism which produces elevated levels of a product on agar will not necessarily do so during production. Furthermore, the intensity of a reporting signal on an agar plate is dependent on the size of a given colony, masking variations in productivity between strains. Agar plate screens usually favor fast-growing strains which tend to give stronger signals.
The analysis of microcolonies has recently been described (Yang et al., Gene 173:19-23 (1996)). This method allows screening of large numbers of colonies on a single agar plate. However, this method still suffers from the problems mentioned above.
More recently, screening of populations of organisms has been performed on microtiter plates. This method generally involves distributing individual clones into wells of microtiter plates, for example by using a colony picker. The cells are then grown in a rich medium which mimics production conditions, allowing cells to grow to a high density. The cells are allowed to grow for various lengths of time. Samples are withdrawn for analysis, frequently requiring dilution of the sample to bring the activity of the target enzyme into the range of the detection method. Activity can be measured by various means, e.g., using chromogenic or fluorogenic substrates, radioactivity, etc.
The microtiter screening methods described above have several disadvantages. They require several liquid handling steps, which limits the throughput and/or requires robotics, which is very expensive. Liquid handling steps are also sources of error. It is also difficult to provide cultures in microtiter plates with oxygen, which is essential for many hosts. Furthermore, the cultures grow to a high density, resulting in changing medium conditions, such as pH, nutrient concentrations, by-products, etc. The changing medium conditions affect the productivity of the cells, resulting in the identification of strains which produce well under such conditions, but not necessarily under production fermentation conditions.
Therefore, there is a need for a rapid and efficient and cost-effective means for screening large numbers of cells for individual strains having preferred productivity of a product under conditions that closely approximate production fermentation conditions. Such a screen would be useful for identifying not only cells of a population with naturally occurring preferred properties, but also for identifying such preferred members of a population resulting from induced mutagenesis.